Myosin VI may be unique among the myosin family members in that it moves toward the pointed (-) end of the actin filament. Furthermore, like myosin V, it is a processive motor that moves via surprisingly large, but variable size, steps along an actin filament. This study has several goals that include, delineating the structural determinants of the reverse directionality, determining the features that allow the large step size as well as the processive movement, and determining the role that calcium plays in the regulation of the motor. This project will utilize in vitro expression and functional assays that will enable structure/function studies of recombinant myosin VI. Expression of enzymatically active fragments (single- and double-headed fragments) of myosin VI will be accomplished with the baculovirus/SF9 cell system. Functional evaluation of the expressed myosin will include ATPase measurements, determination of enzyme kinetic parameters and in vitro motility (translocation of actin filaments by myosin), including single molecule assays (collaboration with Dr. James Spudich). Low-resolution structures of the recombinant myosins bound to actin will be obtained via three-dimensional reconstructions of cryo-electron micrographs derived from decorated actin filaments (collaboration with Dr. Ron Milligan). High-resolution structures of myosin VI in the absence of actin will be obtained by X-ray crystallography (collaboration and subcontract with Dr. Anne Houdusse). The goals of this project will be realized by addressing the following specific aims: 1.) delineate the structural domain(s) in myosin VI that account for myosin VI directionality; 2.) determine which kinetic and structural features are necessary for processive movement and the large step size of myosin VI; and 3) delineate putative modes of regulation of the myosin VI motor.